Compound delivery using impulse transients

ABSTRACT

A method for delivering compounds through epithelial cell layers using impulse transients is described. The method involves applying a compound to, e.g., the stratum corneum, of a patient and then inducing impulse transients to create transient increases in the permeability of epithelial tissue, thereby facilitating delivery of the compound across the epithelial cell layer.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. ProvisionalApplication Ser. No. 60/031,882, filed Nov. 27, 1996, which isincorporated herein in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support from fundingawarded through the Department of Defense Medical Free Electron LaserProgram, N00014-94-I-0927. The Government therefore has rights in theinvention.

BACKGROUND OF THE INVENTION

[0003] This invention relates to the delivery of compounds throughepithelial cell layers using impulse transients, i.e., stress waves.

[0004] Various methods have been employed for facilitating the deliveryof pharmaceutical agents through the skin. One layer of the skin is thestratum corneum, which forms the outermost layer of the epidermis and isthought to act as the skin's primary barrier to molecular transport. Ithas a thickness of 10 to 15 μm and is composed of layers of corneocytes,with the layers varying in thickness from 10 to 50 cells. Corneocytesare keratin-filled cells that lack nuclei and cytoplasmic organelles.Intercellular regions of the stratum corneum are composed mostly ofneutral lipids and comprise 5 to 21% of the stratum corneum volume.

[0005] One method of delivering drugs through the skin is iontophoresis,in which electric current applied to the surface of the skin increasesthe penetration of charged drugs (Singh et al., Med. Re. Rev., 13:569,1993). However, the efficiency of drug delivery using this methoddepends on the ionization state of the drug. In addition, becauseiontophoresis uses high current densities, it can burn the skin (Singhet al., supra).

[0006] In another method, phonophoresis, a drug is delivered throughintact skin using ultrasound (Skauen et al., Intern. J. Pharm., 20:235,1984; Mitragotri et al., J. Pharmaceut. Sci., 84:697, 1995). However,the tensile component of ultrasound waves (negative pressure), which isalways present in ultrasound waves, can cause tissue injury (Ter Haar,Biological Effects of Ultrasound in Clinical Applications, InUltrasound: Its Chemical, Physical, and Biological effects, Suslick,ed., VCH Publishers, pp. 305-20; 1988). In addition, the method requireslong exposure to deliver a therapeutic dose of the drug.

SUMMARY OF THE INVENTION

[0007] The invention is based on the discovery that high pressureimpulse transients, e.g., stress waves (e.g., laser stress waves (LSW)when generated by a laser), with specific rise times and peak stresses(or pressures), can safely and efficiently effect the transport ofcompounds, such as pharmaceutical agents, through layers of epithelialtissues, such as the stratum corneum and mucosal membranes. The newmethods can be used to deliver compounds of a wide range of sizesregardless of their net charge. In addition, impulse transients used inthe methods avoid tissue injury.

[0008] The compounds that can be transported through epithelial tissuelayers by the new methods include pharmaceutical compounds such asphotosensitizers, anesthetic agents, polypeptides, nucleic acids, andantineoplastic agents such as cisplatin, and mixtures of compounds.

[0009] In general, the invention features a method of delivering acompound, e.g., an anesthetic, such as lidocaine, a hormone, such asinsulin, an anti-neoplastic agent, or a nucleic acid, through anepithelial tissue layer by (a) mixing the compound with a couplingmedium to form a compound-coupling medium mixture; (b) contacting asurface of the epithelial tissue layer with the compound-coupling mediummixture; and (c) propagating one or more impulse transients through thecompound-coupling medium mixture to contact and enter the epithelialtissue layer, whereby the compound passes through the epithelial tissuelayer.

[0010] Each impulse transient can be a broad-band compressive wavehaving a rise time of at least 1 ns and a peak pressure of at least 300bar less than that which will damage tissues, e.g., about 2000 bar. Incertain embodiments, the impulse transient can have a duration of about100 ns to 1 microsecond. The impulse transient can be generated byexposing a target material to a pulsed laser beam. The method can beenhanced by adding a step of applying hydrostatic pressure.

[0011] In certain embodiments, a transparent material can be bonded to asurface of the target material to enable confined ablation. In otherembodiments, the target material can be a metallic foil, e.g., ofaluminum or copper, or a plastic sheet, e.g., of a polymer likepolystyrene, and the impulse transient is generated by a laser-inducedplasma formed by ablation of the target material. In another embodiment,the target material can be an absorbing material, and the impulsetransient is generated by laser-induced rapid heating of the absorbingmaterial.

[0012] In another aspect, the invention features an apparatus fordelivering a compound through an epithelial tissue layer. The apparatusincludes a reservoir for containing a coupling medium suitable formixing with the compound, wherein the reservoir is arranged to enablethe coupling medium to directly contact a surface of the epithelialtissue layer; and an energy source, e.g., a laser or lithotripter,arranged and controlled to propagate an impulse transient within thereservoir when filled with the coupling medium.

[0013] In another embodiment, the apparatus further includes a targetmaterial, e.g., a metal foil or plastic sheet, arranged between thelaser and the reservoir, and the reservoir is configured to enable thetarget material to directly contact the coupling material in thereservoir. The apparatus can further include a transparent materialbonded to a surface of the target material and interposed between thesurface and the laser, and arranged to confine pressure forces resultingfrom ablation of the target material within the reservoir. The inventionalso features a system for delivering a compound through an epithelialcell layer in an animal. This system includes the apparatus and acoupling medium suitable for mixing with the compound.

[0014] The laser pulse can have a duration of about 10 to 70 nanoseconds(ns), or in certain embodiments, a duration of about 20 to 40 ns. About1 to 10 laser pulses, and consequently 1 to 10 impulse transients, areapplied to an epithelial cell layer during any one exposure period. Incertain embodiments, about 1 to 3 laser pulses are applied.

[0015] The impulse transients can have a rise time of about 1 to 200 ns.Typically, the impulse transients can have a rise time of about 5 to 15ns.

[0016] The impulse transients can have a peak stress or pressure ofabout 300 to 2000 bars, depending on the nature of the epithelial celllayer. In particular embodiments, the impulse transients can have a peakstress or pressure of about 500 to 1500 bars, e.g., about 550 to 650bars.

[0017] The impulse transients can have a duration of about 100 ns to 1.1microseconds (μs). In specific embodiments, the laser pulse can have aduration of about 150 to about 750 ns, or about 200 to about 300 ns.

[0018] An impulse transient is a broad-band, compressive wave having apeak pressure of up to about 2000 bar, and a fast, but notdiscontinuous, rise time (on the order of 200 ns or less). Accordingly,impulse transients are not shock waves, which are characterized by adiscontinuous rise time. Further, an impulse transient is preferably aunipolar compressive wave, but in addition to the major compressivecomponent, can include a minor tensile component that is less than 5 to10% of the compressive peak pressure.

[0019] A coupling medium is a non-linear liquid or gel medium in whichthe impulse transients are generated and propagated. The coupling mediumenables a direct contact of the impulse transients to the surface of theepithelial cell layer and minimizes acoustic reflections.

[0020] The coupling medium may optionally contain a surfactant toenhance delivery of the compound across the epithelial tissue, e.g., byincreasing the time required for the epithelial tissue to becomeimpermeable following generation of an impulse transient. The surfactantcan be a detergent and thus can include, e.g., sodium lauryl sulfate,cetyl trimethyl ammonium bromide, and lauryl dimethyl amine oxide.

[0021] The invention has many advantages. In particular, the specificrise time and magnitude of the impulse transients used in the newmethods induce a temporary permeability in epithelial tissue layers.This increases the diffusion of compounds through these layers for ashort period of time, and allows effective delivery of the compoundssuch as drugs without causing destruction or killing of cells. Thus, themethod can be used to deliver drugs to desired locations underlyingepithelial cell layers. For example, impulse transients can be used todeliver chemotherapeutic agents to the site of a skin cancer lesion. Inthis manner, a host of maladies can be treated.

[0022] Moreover, drugs that have been previously dismissed because theycould not be transported through epithelial tissue layers, e.g., thestratum corneum layer, can be delivered using the new methods.Similarly, the new methods can also be used to deliver drugs whosetoxicity or high cost precludes or discourages systemic administration.

[0023] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the present document,including definitions, will control. Unless otherwise indicated,materials, methods, and examples described herein are illustrative onlyand not intended to be limiting.

[0024] Various features and advantages of the invention will be apparentfrom the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a graph illustrating the change in fluorescence of skinover time after the addition of 5-aminolevulenic acid (ALA) and a singleimpulse transient to the skin.

[0026]FIG. 2 is a graph illustrating the change in fluorescence of skinover time after the addition of ALA to the skin without an impulsetransient.

[0027]FIG. 3 is a graph illustrating the comparative changes influorescence of skin following the addition of ALA and a single impulsetransient under the indicated peak stresses.

[0028]FIG. 4 is a graph illustrating the waveform of an impulsetransient generated by ablation of a black polystyrene target with asingle 23 nsec Q-switched ruby laser pulse.

[0029]FIGS. 5A and 5B are graphs illustrating the fluorescence spectra(excitation: 486 nm) before generation of an impulse transient(baseline), immediately after exposure to the impulse transient (laserstress wave “LSW”) in the presence of 40 kDa dextran (after LSW) andafter the stratum corneum was removed by tape stripping (“SC removed”)(FIG. 5A; and of the emission spectra of the exposed (+LSW) and control(−LSW) sites after subtraction of baseline fluorescence (FIG. 5B).

[0030]FIG. 6 is a graph illustrating the fluorescence spectra(excitation: 568 nm) of a site exposed to an impulse transient waveusing 20 nm latex particles as the probe material (+LSW) and the controlsite (−LSW).

[0031]FIG. 7 is a graph illustrating the fluorescence spectra(excitation: 568 nm) of two sites exposed to an impulse transient using40 kDA dextran as the probe in the presence (solid line) and absence(dashed line) of a surfactant.

[0032]FIG. 8 is a schematic drawing of a device using hydrostaticpressure to enhance delivery of a compound through the stratum corneumfollowing the application of an impulse transient.

DETAILED DESCRIPTION

[0033] The invention provides new methods for delivering compounds,e.g., pharmaceutical compounds, through multiple cellular layers ofepithelial tissue of a person or animal using impulse transients.Impulse transients induce a transient increase in the permeability ofthe epithelial tissue layer, thereby increasing diffusion of a compoundfrom an exterior region of the epithelial tissue layer, through theepithelial tissue.

[0034] Prior to exposure to an impulse transient, an epithelial tissuelayer, e.g., the stratum corneum or a mucosal layer, is likelyimpermeable to a foreign compound; this prevents diffusion of thecompound into cells underlying the epithelial layer. Exposure of theepithelial layer to the impulse transients enables the compound todiffuse through the epithelial layer. The rate of diffusion, in general,is dictated by the nature of the impulse transients and the size of thecompound to be delivered.

[0035] The rate of penetration through specific epithelial tissue layerssuch as the stratum corneum of the skin also depends on several otherfactors including pH, the metabolism of the cutaneous substrate tissue,pressure differences between the region external to the stratum corneum,and the region internal to the stratum corneum, as well as theanatomical site and physical condition of the skin. In turn, thephysical condition of the skin depends on health, age, sex, race, skincare, and history, for example, prior contacts with organic solvents orsurfactants.

[0036] The amount of compound delivered through the epithelial tissuelayer will also depend on the length of time the epithelial layerremains permeable, and the size of the surface area of the epitheliallayer which is made permeable.

[0037] Properties of Impulse Transients

[0038] The properties and characteristics of impulse transients arecontrolled by the energy source used to create them. However, theircharacteristics are modified by the linear and non-linear properties ofthe coupling medium through which they propagate. The linear attenuationcaused by the coupling medium attenuates predominantly the highfrequency components of the impulse transients. This causes thebandwidth to decrease with a corresponding increase of the rise time ofthe impulse transient. The non-linear properties of the coupling medium,on the other hand, cause the rise time to decrease. The decrease of therise time is the result of the dependence of the sound and particlevelocity on stress (pressure). As the stress increases, the sound andthe particle velocity increase as well. This causes the leading edge ofthe impulse transient to steepen.

[0039] The relative strengths of the linear attenuation, non-linearcoefficient, and the peak stress determine how long the wave has totravel for the rise time steepening to become substantial. This distancecan be calculated from the theory of non-linear acoustics (Lyamshev Sov.Phys. Usp., 24:977, 1981).

[0040] For a planar impulse transient, the distance (L) travelledthrough the coupling medium that leads to non-linear distortions isgiven by equation (1) (Lyamshev Sov. Phys. Usp. 24:977; 1981):$\begin{matrix}{L = \frac{1{pc}^{2}}{ɛ\quad P}} & (1)\end{matrix}$

[0041] where 1 is the spatial width of the rise time (temporal rise timemultiplied by the sound velocity), p the density of the medium, c thesound velocity, ε the non-linear coefficient and P the peak stress orpressure. If the coupling medium is water, for example, an impulsetransient with a temporal rise time of 20 ns and peak pressure of 500bar will show significant steepening within a propagation distance ofabout 1.5 mm.

[0042] The steepening can be calculated from equation (2):$\begin{matrix}{\delta = \frac{pcv}{\varepsilon \quad P}} & (2)\end{matrix}$

[0043] where δ is the width of the rise time and v the dissipativecoefficient (defined by equation 3): $\begin{matrix}{v = \left( \frac{2c^{3}}{\omega \quad P} \right)} & (3)\end{matrix}$

[0044] where ω is the frequency of the peak stress or pressure, and α isthe absorption coefficient at frequency ω.

[0045] The rise time, magnitude, and duration of the impulse transientare chosen to create a non-destructive (i.e., non-shock wave) impulsetransient that temporarily increases the permeability of the epithelialtissue layer. Equations 1, 2, and 3, described above, can be used forcalculating the parameters from published values for different couplingmedia. Generally, the rise time is at least 1 ns, and is more preferablyabout 10 ns.

[0046] The peak stress or pressure of the impulse transients varies fordifferent epithelial tissue or cell layers. For example, to transportcompounds through the stratum corneum, the peak stress or pressure ofthe impulse transient should be set to at least 400 bar; more preferablyat least 1,000 bar, but no more than about 2,000 bar.

[0047] For epithelial mucosal layers, the peak pressure should be set tobetween 300 bar and 800 bar, and is preferably between 300 bar and 600bar.

[0048] The impulse transients preferably have durations on the order ofa few tens of ns, and thus interact with the epithelial tissue for onlya short period of time. Following interaction with the impulsetransient, the epithelial tissue is not permanently damaged, but remainspermeable for up to about three minutes.

[0049] In addition, the new methods involve the application of only afew discrete high amplitude pulses to the patient. The number of impulsetransients administered to the patient is typically less than 100, morepreferably less than 50, and most preferably less than 10. If multipleoptical pulses are used to generate the impulse transient, the timeduration between sequential pulses is 10 to 120 seconds, which is longenough to prevent permanent damage to the epithelial tissue.

[0050] Properties of impulse transients can be measured using methodsstandard in the art. For example, peak stress or pressure, and rise timecan be measured using a polyvinylidene fluoride (PVDF) transducer methodas described in Doukas et al., Ultrasound Med. Biol., 21:961 (1995).

[0051] Generation of Impulse Transients

[0052] Impulse transients can be generated by various energy sources.For example, impulse transients can be generated by ablation orthermoelastic expansion of an appropriate target material by a highenergy optical source such as a laser (Doukas et al., PhysicalCharacteristics and Biological Effects of Laser-Induced Stress Waves,Ultrasound in Med. & Biol., 22:151-164, 1996). When impulse transientsare generated by laser, they can be referred to as laser stress waves.

[0053] The efficiency of conversion of laser energy to mechanical energyof the impulse transient is given by the coupling coefficient of thetarget material. The coupling coefficient (C_(m)) is defined as thetotal momentum transfer to the target material during ablation dividedby the pulse energy. The physical phenomenon responsible for launchingthe impulse transient is, in general, chosen from three differentmechanisms: (1) thermoelastic generation; (2) optical breakdown; or (3)ablation.

[0054] For example, the impulse transients can be initiated by applyinga high energy laser source to ablate a target material, and the impulsetransient is then coupled to an epithelial tissue or cell layer by acoupling medium. The coupling medium can be, for example, a liquid or agel, as long as it is non-linear. Thus, water, oil such as castor oil,an isotonic medium such as phosphate buffered saline (PBS), or a gelsuch as a collagenous gel, can be used as the coupling medium.

[0055] The coupling medium can in addition include a surfactant thatenhances transport, e.g., by prolonging the period of time in which thestratum corneum remains permeable to the compound following thegeneration of an impulse transient. The surfactant can be, e.g., ionicdetergents or nonionic detergents and thus can include, e.g., sodiumlauryl sulfate, cetyl trimethyl ammonium bromide, and lauryl dimethylamine oxide.

[0056] The absorbing target material acts as an optically triggeredtransducer. Following absorption of light, the target material undergoesrapid thermal expansion, or is ablated, to launch an impulse transient.Typically, metal and polymer films have high absorption coefficients inthe visible and ultraviolet spectral regions.

[0057] Many types of materials can be used as the target material inconjunction with a laser beam, provided they fully absorb light at thewavelength of the laser used. The target material can be composed of ametal such as aluminum or copper; a plastic, such as polystyrene, e.g.,black polystyrene; a ceramic; or a highly concentrated dye solution. Thetarget material must have dimensions larger than the cross-sectionalarea of the applied laser energy. In addition, the target material mustbe thicker than the optical penetration depth so that no light strikesthe surface of the skin. The target material must also be sufficientlythick to provide mechanical support. When the target material is made ofa metal, the typical thickness will be {fraction (1/32)} to {fraction(1/16)} inch. For plastic target materials, the thickness will be{fraction (1/16)} to {fraction (1/8)} inch.

[0058] Impulse transients can be also enhanced using confined ablation.In confined ablation, a laser beam-transparent material, such as aquartz optical window, is placed in close contact with the targetmaterial. Confinement of the plasma created by ablating the targetmaterial by using the transparent material increases the couplingcoefficient by an order of magnitude (Fabro et al., J. Appl. Phys.,68:775, 1990). The transparent material can be quartz, glass, ortransparent plastic.

[0059] Since voids between the target material and the confiningtransparent material allow the plasma to expand, and thus decrease themomentum imparted to the target, the transparent material is preferablybonded to the target material using an initially liquid adhesive, suchas carbon-containing epoxies, to prevent such voids.

[0060] The laser beam can be generated by standard optical modulationtechniques known in the art, such as by employing Q-switched ormode-locked lasers using, for example, electro or acousto-optic devices.Standard commercially available lasers that can operate in a pulsed modein the infrared, visible, and/or infrared spectrum include Nd:YAG,Nd:YLF, CO₂, excimer, dye, Ti:sapphire, diode, holmium (and otherrare-earth materials), and metal-vapor lasers. The pulse widths of theselight sources are adjustable, and can vary from several tens ofpicoseconds (ps) to several hundred microseconds. For use in the newmethods, the optical pulse width can vary from 100 ps to about 200 nsand is preferably between about 500 ps and 40 ns.

[0061] Impulse transients can also be generated by extracorporeallithotripters (one example is described in Coleman et al., UltrasoundMed. Biol., 15:213-227, 1989). These impulse transients have rise timesof 30 to 450 ns, which is longer than laser-generated impulsetransients. To form an impulse transient of the appropriate rise timefor the new methods using an extracorporeal lithotripter, the impulsetransient is propagated in a non-linear coupling medium (e.g., water)for a distance determined by equation (1), above. For example, whenusing a lithotripter creating an impulse transient having a rise time of100 ns and a peak pressure of 500 barr, the distance that the impulsetransient should travel through the coupling medium before contacting anepithelial cell layer is approximately 5 millimeters (mm).

[0062] An additional advantage of this approach for shaping impulsetransients generated by lithotripters is that the tensile component ofthe wave will be broadened and attenuated as a result of propagatingthrough the non-linear coupling medium. This propagation distance shouldbe adjusted to produce an impulse transient having a tensile componentthat has a pressure of only about 5 to 10% of the peak pressure of thecompressive component of the wave. Thus, the shaped impulse transientwill not damage tissue.

[0063] The type of lithotripter used is not critical. Either aelectrohydraulic, electromagnetic, or piezoelectric lithotripter can beused.

[0064] The impulse transients can also be generated using transducers,such as piezoelectric transducers. Preferably, the transducer is indirect contact with the coupling medium, and undergoes rapiddisplacement following application of an optical, thermal, or electricfield to generate the impulse transient. For example, dielectricbreakdown can be used, and is typically induced by a high-voltage sparkor piezoelectric transducer (similar to those used in certainextracorporeal lithotripters, Coleman et al., Ultrasound Med. Biol.,15:213-227, 1989). In the case of a piezoelectric transducer, thetransducer undergoes rapid expansion following application of anelectrical field to cause a rapid displacement in the coupling medium.

[0065] In addition, impulse transients can be generated with the aid offiber optics. Fiber optic delivery systems are particularly maneuverableand can be used to irradiate target materials located adjacentepithelial tissue layers to generate impulse transients in hard-to-reachplaces. These types of delivery systems, when optically coupled tolasers, are preferred as they can be integrated into catheters andrelated flexible devices, and used to irradiate most organs in the humanbody. In addition, to launch an impulse transient having the desiredrise times and peak stress, the wavelength of the optical source can beeasily tailored to generate the appropriate absorption in a particulartarget material.

[0066] Delivery of Compounds Using Impulse Transients

[0067] Because impulse transients exert physical forces to increase thepermeability of the epithelial tissue, they can be used to transportmany different types of compounds. Thus, chemotherapeutic agents such ascisplatin, polypeptides, such as antibodies, nucleic acids, such asoligonucleotides, DNA, RNA, and plasmids, local anesthetics, such aslidocaine and benzocaine, and photosensitizers, such as benzoporpherenederivative monoacid ring A (BPD-MA), all can be delivered throughepithelial tissue layers, e.g., transdermally, using impulse transients.The compounds may optionally be heated prior to generation of theimpulse transient to facilitate their transport through the skin.

[0068] Localization of the compound using the methods of the inventionis advantageous, as it allows impulse transients to be administered withhighly localized effects to areas of diseased cells, thus sparing theother tissues of the body. In this way, healthy tissues and organs arespared from adverse effects of a systemically administered drug.

[0069] Compounds which have a toxic effect at higher dosages can beadministered to a patient using guidelines for administration that willproduce greater concentrations of the drugs in the treated tissues orcells compared to the surrounding tissues, while maintaining adequatelevels of the drug in these treated tissues or cells. In general, thisdifferential drug localization can be achieved using guidelines foradministration determined using standard techniques known in the fieldof pharmacology. Preferably, the compound dosage and time course aresuch that a 2:1 or greater concentration ratio is achieved in thetreated tissues or cells compared to the surrounding, untreated tissues.

[0070] Determining the appropriate dosage for a specific compound, andfor a particular subject or patient (human or animal) is a routinematter to one skilled in the art of pharmaceutical administration. Twoapproaches are commonly used to assay directly the quantity of drug inthe diseased (treated) and surrounding tissues. First, tissue samplesare obtained from animals (e.g., pigs) or patients who have been treatedwith different dosage and timing protocols. Cadaver skin samples canalso be used in this assay. The quantity of drug in each tissue is thenmeasured either chemically, or if there is a unique optical signal suchas fluorescence, then by quantitative microscopy or laser-inducedfluorescence. The results are tabulated to determine a scale of optimumdrug dosages and types of impulse transients for a given epithelialtissue layer, body region, and compound.

[0071] The compound or compounds to be delivered through an epithelialcell layer are administered by mixing the compound with the couplingmedium, and applying the coupling medium-compound mixture to the surfaceof the epithelial cell layer, e.g., the stratum corneum, in the regionin which transport is desired. The compound must be thoroughly dispersedin, and is preferably dissolved in, the coupling medium. Thus,hydrophilic compounds can be mixed with an aqueous coupling medium, andhydrophobic compounds can be mixed with an oil-based coupling medium.

[0072] Once the target material and coupling medium in a container areset in position on a particular region of the surface of an epithelialtissue layer, impulse transients are used to permeabilize the epithelialtissue layer in the region in which the coupling medium directlycontacts the cell layer, using the methods described herein. The methodsresult in the delivery of the compounds to the cells underlying theepithelial tissue layer in the region of interest that normally wouldnot cross the epithelial tissue layer barrier.

[0073] Hydrostatic pressure can be used in conjunction with impulsetransients to enhance the transport of a compound through the epithelialtissue layer. Since the effects induced by the impulse transients lastfor several minutes, the transport rate of a drug diffusing passivelythrough the epithelial cell layer along its concentration gradient canbe increased by applying hydrostatic pressure on the surface of theepithelial tissue layer, e.g., the stratum corneum of the skin,following application of the impulse transient. This method is describedin further detail in the examples below. The hydrostatic medium can beany liquid, such as water or phosphate buffered saline.

[0074] Topical application and delivery of compounds by the new methodsallow the compounds to be localized to a site of interest. Thus, thecompound, e.g., a drug, is more concentrated at the site of action andhas a minimal, if any, systemic concentration. This enhances thetherapeutic effect of the drug and simultaneously minimizes systemicside-effects. Another advantage compared to systemic administration isthat compounds transported through epithelial tissue bypass systemicdeactivation or degradation (e.g., hepatic “first-pass” effects).Gastrointestinal incompatibility and potential toxicological risks arealso minimized relative to systemic administration. In addition, drugsdeveloped for topical application can be designed so that they aredeactivated systematically (i.e., the “soft drug” concept), usingstandard techniques. Topical administration may also be desired when thecompound is rare or expensive.

EXAMPLES

[0075] The following examples are used to describe the delivery ofcompounds using impulse transients.

Example 1 Transdermal Delivery of ALA

[0076] 5-aminolevulenic acid (ALA) was used as a compound to demonstratethe permeation effect of impulse transients on the stratum corneum. ALAis converted in cells to protoporphyrin IX, which fluoresces at 634 nm(405 nm excitation), while ALA does not fluoresce. Thus, the transportof ALA can be followed, non-invasively, by monitoring the fluorescenceof the skin. In addition, since the conversion of ALA to protophyrin IXrequires that cells be viable, the measurement of protophyrin IXfluorescence also assays cell viability in vivo.

[0077] For these experiments, a Q-switched solid state ruby laser (20 nspulse duration, capable of generating up to 2 joules per pulse) was usedto generate the laser beam, which hit the target material (blackpolystyrene sheet about 1 mm thick) and generated a single impulsetransient. Impulse transients of up to 1000 bar peak stress and 50 nsduration and with a {fraction (1/2)} inch beam diameter can be generatedwith this laser-target system. The large target ensures that the impulsetransients generated are plane waves, because the thickness of thecoupling medium is much shorter than the diameter of the impulsetransient. An articulating arm was used and the laser path was totallycovered for safety.

[0078] A plastic (flexible) washer approximately 1 inch in diameter and{fraction (1/16)} inches thick was used as a reservoir for the samplesolution (5% concentration of the ALA in PBS coupling medium) to bedelivered through the stratum corneum. The washer was attached onto theskin with grease. The sample filled the central opening of the washer,which was approximately {fraction (1/4)} inch in diameter. The targetmaterial was positioned on top of the washer and irradiated with 1 laserpulse.

[0079] The black polystyrene target completely absorbed the laserradiation so that the skin was exposed only to impulse transients, andnot laser radiation. The impulse transients, even at the highest peakstress of 1,000 bar, did not produce any pain in the subject. Afterexposure to the impulse transients, the excess ALA solution was removed.The skin was monitored for fluorescence of protophyrin IX thirty minutesafter exposure to the impulse transients. The fluorescence intensityincreased for approximately 4 hours, at which point it reached themaximum intensity and subsequently decreased.

[0080]FIG. 1 shows the change of fluorescence intensity at differentwavelengths over time after the application of a single impulsetransient. As shown in the graph, the peak in intensity occurs at about640 nm and is highest after 210 minutes (dashed line) post-treatment.

[0081]FIG. 2 shows the fluorescence from an adjacent site (control)where ALA was applied without any impulse transients. As shown in thisgraph, there is little change in the intensity at different time points.

[0082]FIG. 3 shows the effects of varying the applied peak stress of theimpulse transient on ALA transport. As shown in the graph, the degree ofpermeabilization of the stratum corneum depends on the peak stress. Inthree separate experiments, a single impulse transient was applied at500 mJ, 600 mJ, or 1 J to generate applied peak stresses of 300 bar, 400bar, and 600 bar, respectively. FIG. 3 shows that protophyrin IXfluorescence increased with increasing peak pressure, demonstrating thattransdermal transport of ALA increases with increasing peak stress. Theonset of the permeabilization of the stratum corneum was observed above300 bar.

[0083] The permeabilization of the stratum corneum is transient. Whensites on the stratum corneum were exposed first to impulse transientsand ALA was then applied on the same sites after 5 minutes, nofluorescence emission from protophyrin IX was observed. Therefore, thepermeabilization of the stratum corneum lasted less than 5 minutes.

[0084] Penetration of ALA through the skin (without the action ofimpulse transients) depends on many conditions, such as skin hydration,skin temperature, anatomical site, condition of skin and contact time.All fluorescence measurements were compared to the fluorescence emissionof the target site on the stratum corneum before the experiments.

Example 2 Transdermal Delivery of 40 kDa Dextran and 20 nm LatexParticles

[0085] The ability of impulse transients to deliver large macromoleculesacross the stratum corneum was determined using probes of rhodamine Bdextran having a molecular weight of 40 kDa and a diameter of about 8.8nm, and fluorescent latex particles 20 nm in diameter.

[0086] Ten-week old male fuzzy rats, each having a mass of 300-400 g,were obtained from Harlan-Sprague-Dawley (Indianapolis, Ind.) andacclimated for a minimum of 48 hours prior to use. Animals wereanesthetized by intramuscular injection of ketamine (120 mg/kg),xylazine (20 mg/kg), and atropine (0.04 mg/kg).

[0087] A single laser pulse was delivered to the target material, whichgenerated a single impulse transient. Aqueous probe solutions of 500 μMrhodamine B dextran of 40 kDA molecular weight (Molecular Probes,Eugene, Oreg.) or 2% (weight/volume) fluorescent latex particles, 20 nmin diameter (Molecular Probes, Eugene, Oreg.) were allowed to remain incontact with the skin for five minutes after the application of theimpulse transient. Subsequently, the solution was removed and thesurface of the skin was cleaned with water. Control sites adjacent tothe sites exposed to impulse transients were treated with the donorsolution in an identical manner except that they were not exposed toimpulse transients. In addition, some control sites were exposed to aimpulse transient using sterile water only as the coupling medium.

[0088] A flexible washer approximately 19 mm in diameter was used as areservoir for the donor solution to be delivered through the stratumcorneum. The washer was attached on the skin on the dorsal side of eachrat with grease, and a black polystyrene target material was placed ontop of the washer in contact with the solution. The solution acted asthe acoustic coupling medium.

[0089] Impulse transients were generated by ablation of the targetmaterial (Perri, Phys. Fluids 16:1435-1440, 1973) with a 23 nsec pulsefrom a Q-switched ruby laser and launched into the reservoir containingthe molecular probe solution. An articulated arm was used to deliver thebeam to the target. The beam size at the target was about 6 mm indiameter to achieve a fluence of about 7 J/cm². The laser pulse wascompletely absorbed by the target so that only the impulse transientpropagated through the probe solution and impinged onto the skin of therat. The impulse transients were measured in separate experiments underidentical conditions of laser parameters, target material, andpropagation distance through the coupling medium with a calibratedpolyvinylidene fluoride transducer (Doukas et al. Ultrasound Med. Biol.21:961-967, 1995).

[0090] The temporal profile of the impulse transients used in theseexperiments is shown in FIG. 4. The peak stress in the skin (P_(s)) wascalculated from the peak pressure in water (P_(w)) and the acousticimpedance of water (Z_(w)=1.48×10⁶ kgm⁻²s⁻¹) and skin (Z_(s)=1.54×10⁶kgm⁻²s⁻¹) (Payne et al., Sound Skin Models-Acoustic Properties ofEpidermis and Dermis. In Skin Models To Study Function and Disease ofSkin. Parks et al., ed., Springer Verlag, Berlin, pp. 402-411; 1986)using the equation P_(s)/P_(w)=2Z_(w)/(Z_(s)Z_(w)). The peak stress onthe skin in all experiments was calculated to be 589±23 bar.

[0091] The delivery of the dextran and latex beads across the stratumcorneum following the generation of impulse transients was observedusing transmission photomicrographs and fluorescence emission spectra ofbiopsy samples. For these studies, skin samples were obtained one hourpost-treatment using a 6 mm biopsy punch. Biopsies were embedded in OCT4583™ (Sakura Finetek USA, Torrence, Calif.) and frozen. The skinsamples were then sectioned in a cryostat microtome, andmicrophotographs were obtained with a Zeiss inverted microscope using aRhodamine B filter set (XF39, Omega Optical, Brattleboro, Vt.).Fluorescence emission spectra of the exposed and control sites werecollected from another group of animals while they were alive and underfull anesthesia using a fiber-based fluorimeter (FLUORMAX™, SpexIndustries, Edison, N.J.).

[0092] Transmission photomicrography revealed that rhodamine B dextranpenetrated to a depth of approximately 50 μm into the skin. Fluorescencespectra also demonstrated that the rhodamine dextran penetrated thestratum corneum following induction of an impulse transient. Thefluorescence emission spectra of skin exposed to a single impulsetransient in the presence of 40 kDa dextran is shown in FIG. 5A.Emission spectra were taken at three different times: (1) beforeapplication of the dextran probe and generation of the impulsetransient, in order to establish the baseline fluorescence (shown as thedashed line marked “baseline”), (2) immediately after generation of theimpulse transient (shown as the broken line labeled “after LSW,” forlaser stress wave), and (3) after the stratum corneum of the exposedsite was removed by tape stripping (shown as the solid line marked “SCremoved”). Tape stripping was performed to eliminate the fluorescencefrom the probe molecules located in the stratum corneum. Thus, thefluorescence signal in the tape stripping experiment represented onlythe probe molecules located in the viable epidermis and dermis. Twentytape strippings were sufficient to remove the stratum corneum (Wells,Br. J. Dermatol. 108:87-91, 1957). The data shown in FIG. 5A representraw fluorescence.

[0093] The spectra shown in FIG. 5A indicate that rhodamine-associatedfluorescence increased following delivery of an impulse transient toskin exposed to the 40 kDa rhodamine dextran probe (spectra labeled“after LSW”). Most of this fluorescence remained after removal of thestratum corneum (compare the spectra labeled “SC removed” with “afterLSW”). Both of these spectra show significantly higher intensity thanthat shown by the baseline spectra. In addition, exposure of skin toimpulse transients only did not induce any change in the fluorescenceemission of the skin (data not shown). These data suggest thatapplication of an impulse transient (in the form of a laser stress wave)caused the 40 kDa rhodamine dextran probe to be transported into thedermis, i.e., to be localized in tissues that are not sensitive toprocedures that remove the stratum corneum.

[0094]FIG. 5B shows the comparative fluorescence of sites exposed to theLSW (+LSW) and control sites (−LSW) after tape stripping and after thebaseline fluorescence has been subtracted. The site subjected to animpulse transient showed over two-fold higher rhodamine associatedfluorescence than the control site, which also demonstrates that impulsetransients promote transport of the dextran probe across the stratumcorneum.

[0095] Latex fluorescent particles of 10 nm diameter were also deliveredthrough the stratum corneum using impulse transients. FIG. 6 shows thefluorescence emission spectra after tape stripping of a site exposed toa impulse transient using latex particles as the fluorescent probe. Thefluorescence emission of the control site under identical conditions isshown for comparison.

[0096] To measure the amount of time required for the stratum corneum toregain its barrier function for a probe molecule having the size of 40kDa dextran, a single impulse transient was applied to skin usingsterile water as the acoustic coupling medium. The coupling medium wasimmediately removed, and the probe solution added to the reservoir 2minutes after the application of the impulse transient. The fluorescenceemission spectra were then measured as described above. No fluorescencewas detected. This indicates that the stratum corneum becomesimpermeable to 40 kDa dextran within 2 minutes after the generation ofthe impulse transient.

Example 3 Transdermal Delivery Using Surfactants in the Coupling Medium

[0097] The effect of a surfactant was examined by using a solution of 2%sodium lauryl sulfate (SLS) as the coupling medium. FIG. 7 shows thefluorescence spectra (from which baseline fluorescence had beensubtracted) of the 40 kDa dextran probe molecule in skin at two sites atwhich an impulse transient had been generated. At one site (solid line),2% SLS was used as the coupling medium; at the other, no SLS was added(dashed line). The two skin sites were tape stripped prior to generatingthe two spectra shown in FIG. 7.

[0098] A comparison of the spectra shown in FIG. 7 reveals that thefluorescence intensity of 40 kDa dextran was approximately 8-9 foldgreater when 2% SLS was used in the coupling medium. Thus, a surfactantcan significantly increase the amount of an agent delivered across thestratum corneum using an impulse transient.

[0099] To determine if surfactants in the coupling medium affecttransport by increasing the time of recovery of the barrier function ofthe stratum corneum, the length of time to restore the barrier functionof the stratum corneum was compared using water or an aqueous solutionof 2% sodium lauryl sulfate (SLS) as the coupling medium.

[0100] As discussed above in Example 2, the stratum corneum becomesimpermeable to the 40 kDa dextran probe within two minutes aftergeneration of an impulse transient. To determine the length of time thestratum corneum remains permeable when impulse transients are generatedin the presence of a surfactant, a single pulse was applied in which theinitial coupling medium was an aqueous solution of 2% SLS. Thesurfactant was removed, and the aqueous solution of the 40 kDa dextranprobe was added 15, 30, 45, and 60 minutes after generation of theimpulse transient. The presence of the probe was then measured.

[0101] Probe molecules added as long as 45 to 60 minutes aftergeneration of the impulse transient emitted fluorescence that wasresistant to procedures that remove the stratum corneum. Theseobservations indicate that when the surfactant was used in the couplingmedium, the recovery of the barrier function of the stratum increased to45-60 minutes. This compares to recovery of the barrier function within2 minutes without the surfactant. Surfactants therefore can act toincrease the time required for the stratum corneum to regain its barrierfunction.

Example 4 Transdermal Delivery of Anti-neoplastic Agents

[0102] 5-fluorouracil (5-FU) is dissolved in an aqueous solution of PBS,which serves as the coupling medium, and applied to a skin cancer lesionin a suitable container as described in Example 1. A black polystyrenesheet is used as the target material and is placed on the container indirect contact with the coupling medium, which in turn transmits theimpulse transients to the surface of the lesion. Five pulses from aQ-switched solid state ruby laser (1 J, 20 ns pulse duration) areapplied to generate 5 separate impulse transients. The amount of 5-FUdissolved in the PBS is determined using standard techniques describedherein and based on the nature of the lesion, the desired drugconcentration in the lesion, and the patient's skin type.

Example 5 Transdermal Delivery of ALA Using Impulse Transients andConfined Ablation

[0103] In confined ablation, a transparent material is placed in closecontact with the target material. Confinement of the plasma increasesthe coupling coefficient by an order of magnitude.

[0104] A quartz optical window with a thickness of {fraction (3/8)} inchis used as the transparent material, and a black polystyrene sheet isused as the target material. To eliminate microscopic voids, a solventis used to dissolve the surface of the polystyrene to allow it to bondto the quartz transparent material. This combined transparent materialand target material is used in the same way as the target material inExample 1.

[0105] Plasma confinement causes an increase in the rise time of theimpulse transient, which may decrease the effectiveness of the impulsetransient. To counteract this effect, the distance the impulse transientpropagates through the coupling medium is increased to shape the impulsetransient to have an appropriate rise time when it contacts the surfaceof the epithelial tissue layer. The non-linear properties of thecoupling medium cause the rise time to decrease. An impulse transient of500 bar propagating through 1 mm of water undergoes a decrease in therise time from 30 ns to 15 ns. Therefore, the initial increase of therise time due to confinement can be compensated by appropriatelyadjusting the propagation length of the impulse transient through thereservoir.

[0106] The impulse transient is measured in separate experiments underidentical conditions using a calibrated polyvinylidene fluoridetransducer as described in Doukas et al., Ultrasound Med. Biol., 21:961(1995). The temporal resolution of the combination of the transducer andoscilloscope is 5 ns. The pressure in the skin P_(s) is calculated fromthe pressure in water P_(w) and the acoustic impedances of water (Z_(w))and skin (Z_(s)) using the equation P_(s)/P_(w)=2 Z_(w)/(Z_(s)+Z_(w)).

[0107] Impulse transients are generated by using one pulse of aQ-switched ruby laser (4 J, 30 ns). An ArF excimer laser can also beused (650 mJ, 25 ns). Transcutaneous delivery of ALA is measured bymeasuring protophyrin IX fluorescence as described above.

Example 6 Transdermal Delivery of Benzoporpherene Derivative MonoacidRing A (BPD-MA) Using Impulse Transients and Hydrostatic Pressure

[0108]FIG. 8 shows a schematic diagram of a device 10 that is used tocontrol drug delivery by varying the hydrostatic pressure. A reservoir12 is made from a plastic washer 14 about 3 mm in height and 1 cm indiameter and is attached to rabbit skin 16 with silicon grease. Anoutlet 20 that connects to a groove 18 in the bottom of the washer isconnected to a suction pump (not shown). This allows the washer toremain firmly vacuum sealed on the skin during the application ofhydrostatic pressure. A black polystyrene target material 22 is attachedto the surface of the washer 14, and a quartz overlay 23 is placed onthe target material. Laser radiation 24 from Q-switched ruby laser (4 J,30 ns) 26 is directed onto the target material 22 using an articulatingarm (not shown). A tube 28 is connected to an opening 21 in the side ofthe washer 14. The tube 28 is connected to a pressure regulator 30, andthe reservoir 12 is filled with a solution of benzoporpherene derivativemonoacid ring A (BPD-MA).

[0109] The fur on the back of a New Zealand albino rabbit is removed.The animal is anesthetized and the device 10 for applying hydrostaticpressure is attached to the back of the animal. Subsequently,hydrostatic pressure is applied and a single impulse transient isgenerated. The hydrostatic pressure is applied for 5 minutes. The deviceis then removed, the skin cleaned, and the fluorescence is measuredusing a fiber-based spectrofluorimeter (450 nm excitation, 650-750 nmemission).

[0110] These measurements are compared to three control sites in whichBPD-MA is applied. One site is exposed to a single impulse transientwith no hydrostatic pressure applied. The second is exposed only tohydrostatic pressure and no impulse transient, and the third is exposedto neither an impulse transient nor hydrostatic pressure.

[0111] The fluorescence measurements indicate the amount of BPD-MApresent in the skin. To confirm these measurements, BPD-MA is extractedfrom the tissue and measured using standard techniques. Briefly, skinbiopsies are weighed, mixed with 1 ml dimethyl sulfoxide (DMSO) andhomogenized. The homogenized samples are kept at room temperatureovernight and then centrifuged. The integrated fluorescence of thesupernatant is measured in a spectrofluorimeter and the amount of drugis estimated from a calibration curve.

Example 7 Transdermal Delivery of a Compound Using a Lithotripter

[0112] A subject places a limb having the desired target area of skininto a water bath. The target area is covered by a small reservoircontaining a 5% ALA solution in PBS. This reservoir differs from othersdescribed above in that it is covered on the side opposite the skin witha thin plastic film or membrane that has an impedance near that ofwater, i.e., it is designed not to reflect impulse transients generatedby the lithotripter that propagate first through the water and thenthrough the reservoir to reach the target epithelial layer. The ALA-PBSsolution in the reservoir serves as both a source of the compound (ALA)and the coupling medium.

[0113] Ten pulses from a electrohydraulic lithotripter are applied inthe water bath to generate ten impulse transients. The rise times andpeak stress are adjusted to be about 5 to 15 ns and 500 bar,respectively, at the point of contact with the skin, followingpropagation through the water bath and the coupling medium.

[0114] Transdermal delivery of ALA is determined by measuringprotophyrin IX fluorescence as described above.

Other Embodiments

[0115] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, that theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. An apparatus for delivering a compound through anepithelial tissue layer, the apparatus comprising a reservoir forcontaining a coupling medium suitable for mixing with the compound,wherein the reservoir is arranged to enable the coupling medium todirectly contact a surface of the epithelial tissue layer; and an energysource arranged and controlled to propagate an impulse transient withinthe coupling medium when in the reservoir.
 2. An apparatus of claim 1,wherein the energy source is a laser, and the apparatus furthercomprises a target material arranged between the laser and thereservoir, and wherein the reservoir is configured to enable the targetmaterial to directly contact the coupling material in the reservoir. 3.An apparatus of claim 2, wherein the target material is a metal foil orplastic sheet.
 4. An apparatus of claim 1, further comprising atransparent material bonded to a surface of the target material andinterposed between the surface and the laser, and arranged to confinepressure forces resulting from ablation of the target material withinthe reservoir.
 5. An apparatus of claim 1, wherein the energy source isa lithotriptor.
 6. An apparatus of claim 3, wherein the metal foilcomprises aluminum or copper.
 7. An apparatus of claim 2, wherein thetarget material comprises a polymer.
 8. A system for delivering acompound through an epithelial cell layer in an animal, the systemcomprising an apparatus of claim 1; and a coupling medium suitable formixing with the compound.
 9. A method of delivering a compound throughan epithelial tissue layer, the method comprising: (a) mixing thecompound with a coupling medium to form a compound-coupling mediummixture; (b) contacting a surface of the epithelial tissue layer withthe compound-coupling medium mixture; and (c) propagating one or moreimpulse transients through the compound-coupling medium mixture tocontact and enter the epithelial tissue layer, whereby the compoundpasses through the epithelial tissue layer.
 10. A method of claim 9,wherein each impulse transient is a broad-band compressive wave having arise time of at least 1 ns and a peak pressure of at least 300 bar andno more than 2000 bar.
 11. A method of claim 9, wherein the impulsetransient is generated by exposing a target material to a pulsed laserbeam.
 12. The method of claim 11 wherein a transparent material isbonded to a surface of the target material.
 13. A method of claim 9,wherein the compound is a nucleic acid.
 14. A method of claim 9, whereinthe compound is an anti-neoplastic agent.
 15. The method of claim 11,wherein the target material comprises a metallic foil or a plasticsheet, and wherein the impulse transient is generated by a laser-inducedplasma formed by ablation of the target material.
 16. The method ofclaim 15, wherein the metallic foil comprises aluminum or copper. 17.The method of claim 11, wherein the target material comprises a polymer.18. The method of claim 11, wherein the target material comprises anabsorbing material, and wherein the impulse transient is generated bylaser-induced rapid heating of said absorbing material.
 19. A method ofclaim 9, further comprising a step of applying hydrostatic pressure. 20.A method of claim 9, wherein the epithelial tissue layer is stratumcorneum.
 21. A method of claim 9, wherein said coupling medium furthercomprises a surfactant.
 22. A method of claim 21, wherein saidsurfactant is sodium lauryl sulfate.
 23. A method of claim 11, whereinthe impulse transient has a peak pressure of 550-650 bar.
 24. A methodof claim 11, wherein the impulse transient has a rise time of about75-125 ns.